Methods and compositions for controlled release of bioactive compounds

ABSTRACT

A controlled release pharmaceutical dosage form for bacteriophage is presented. The dosage form is prepared by drying the active ingredient together with a dispersion or solution of polymethacrylate copolymer and a lyprotectant using mild, entirely aqueous conditions.

PRIOR APPLICATION INFORMATION

[0001] This application claims priority on U.S. S No. 60/463,319, filedApr. 17, 2003.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of drugdelivery.

BACKGROUND OF THE INVENTION

[0003] Bacterial infections in carrier animals by human pathogens, suchas Escherichia coli O157:H7, Campylobacter spp., and Salmonella spp.,etc, may be controlled by bacteriophages that attack and kill thesebacteria. In order for enteric infections to be controlled withbacteriophages the bacteriophages must be given to animals by mouth in aform that will allow them to act at the site of infection, typicallydistal to the stomach. Bacteriophage have properties that makepreparation of suitable dosage forms difficult. For example,bacteriophages are typically damaged irreversibly by exposure to harshconditions, by conventional drying techniques and by low pH andproteolysis in the stomach and in the gastrointestinal tract. Thisdamage results from pH-dependent denaturation and proteolytic degrationof proteins vital to the viability of the bacteriophage. Bacteriophagerepresent extremely complicated therapeutics since they are comprised ofDNA, or RNA and proteins. Frequently, bacteriophages contain elaboratereceptor-binding tail structures that are particularly sensitive tobreakage. In essence, our ability to exploit the therapeutic potentialof bacteriophages is severely limited by the sensitivity ofbacteriophages to low pH, proteolytic degradation and denaturation dueto drying, and the lack of suitable methods for preparing economical andeffective solid dosage forms.

[0004] The therapeutic potential of bacteriophage is described invarious reports. In these studies the bacteriophage were used in aqueousform with or without buffers to neutralize stomach acid. Theseformulations are not ideal because they lack practicality. No methods toproduce stabilized, controlled release dosage formulations ofbacteriophage have been described.

[0005] A bacteriophage delivery system is needed deliver bacteriophagein a controlled manner to enable commercial development of the agents.

[0006] Methacrylic acid copolymers (Eudragits) have been usedextensively to prepare controlled release oral dosage forms of drugs.These polymers have great utility for this purpose because they havebeen engineered to become soluble in different pH environments. Forexample, Eudragit L100 dissolves above pH 5.5 and will protect an activeingredient in acid environments, such as the stomach, yet upon exposureto neutral or basic environments the same dosage form will release theactive ingredient. In this regard, the aqueous solubility of themethacrylic acid copolymers is controlled by the degree of protonationof carboxyl groups, which are present on the polymer backbone. If thecarboxyl groups are deprotonated, as occurs in basic or neutralenvironments, the resulting ionic carboxylate groups increase theaqueous solubility of the polymer.

[0007] Methacrylic acid copolymers are typically applied todrug-containing cores as thin layers by ladling or spray coating.Alternatively, they may be incorporated into monolithic matrix devicesby compression techniques, or by other techniques, such as spray drying.Methacrylic acid copolymers are used generally in the protonated form,either as aqueous dispersions under low pH conditions, or as solutionsin organic solvents. Using these conditions has a number of drawbacks.For example, the physical characteristics of the material in dispersionmay be detrimental to the drug activity, particularly when it is abiological macromolecule. Similarly, the activity of the drug may beadversely affected by exposure to organic solvents.

[0008] Methacrylic acid copolymers have not been used to coatbacteriophages for enteric delivery.

SUMMARY OF THE INVENTION

[0009] According to a first aspect of the invention, there is provided apharmaceutical composition comprising:

[0010] a methacrylic acid polymer solution or dispersion;

[0011] a lyoprotectant; and

[0012] a bioactive agent.

[0013] According to a second aspect of the invention, there is provideda method of preparing a pharmaceutical composition comprising:

[0014] mixing a methacrylate polymer and water;

[0015] adding a lyoprotectant and a bioactive agent to the mixture; and

[0016] drying the mixture.

[0017] According to a third aspect of the invention, there is provided amethod of treating a bacterial infection comprising:

[0018] administering to an animal in need of such treatment atherapeutic amount of a dried pharmaceutical composition comprising:

[0019] a methacrylic acid polymer;

[0020] a lyoprotectant; and

[0021] a bacteriophage.

[0022] According to a fourth aspect of the invention, there is provideda method of treating a bacterial infection comprising:

[0023] administering to an animal in need of such treatment atherapeutic amount of a dried pharmaceutical composition comprising:

[0024] a methacrylic acid polymer;

[0025] a lyoprotectant; and

[0026] a bioactive agent.

[0027] According to a fifth aspect of the invention, there is provided amethod of treating a disease condition comprising:

[0028] administering to an animal in need of such treatment atherapeutic amount of a dried pharmaceutical composition comprising:

[0029] a methacrylic acid polymer;

[0030] a lyoprotectant; and

[0031] a bioactive agent.

BRIEF DESCRIPTION OF THE TABLES

[0032] Table 1. Viability of rV5 in formulations containing a)polymethacrylate copolymer (Eudragit L100), b) sucrose, and c)polymethacrylate copolymer (Eudragit L100) and sucrose.

[0033] Table 2. Viability of rV5 in formulations containing a)polymethacrylate copolymer (Eudragit L100), b) sucrose, and c)polymethacrylate copolymer (Eudragit L100) and sucrose.

[0034] Table 3. Protection of rV5 to acid treatment for 20 minutes byformulations containing a) polymethacrylate copolymer (Eudragit L100),b) sucrose, and c) polymethacrylate copolymer (Eudragit L100) andsucrose.

[0035] Table 4. Protection of rV5 to acid treatment overnight byformulations containing a) polymethacrylate copolymer (Eudragit L100),b) sucrose, and c) polymethacrylate copolymer (Eudragit L100) andsucrose.

[0036] Table 5. Controlled release of bacteriophage rV5 from formulationmade using excipient containing 5% sucrose and 10% polymethacrylateS100.

[0037] Table 6. Controlled release of bacteriophage rV5 from formulationmade using excipient containing 5% sucrose and 5% polymethacrylate S100.

[0038] Table 7. Controlled release of bacteriophage rV5 and wV8 fromformulations containing polymethacrylate copolymer (Eudragit S100) andsucrose.

[0039] Table 8. Rapid release of rV5 and wV8 from formulationscontaining polymethacrylate copolymer (Eudragit S100) and sucrose.

[0040] Table 9. Viability of bacteriophage rV5 in formulationscontaining polymethacrylate copolymer (Eudragit S100) and sucroseprepared by spray drying.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedhereunder are incorporated herein by reference.

[0042] Definitions

[0043] As used herein, “bioactive compound” or “bioactive agent” refersto compounds having a biological effect. Examples include but are by nomeans limited to bacteriophage, pharmaceutical compounds, antibodies,receptor ligands, viruses, peptides, peptide fragments and the like.

[0044] As used herein, “animals” refers to vertebrates andinvertebrates.

[0045] As used herein, “enteropathic organism” refers to an organismcapable of colonizing the gastrointestinal tract of an animal. Examplesof enterotoxigenic microogranisms include but are by no means limited toBacillus cereus, Bacillus anthracis, Bacillus subtilis, Bacillusthuringiensis, Bacillus stearothermophilus, Vibrio parahemolyticus,Vibrio cholerae O1, Vibrio cholerae non-O1, Vibrio vulnificus,Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonellaentertidis, Salmonella cholerasuis, Salmonella typhimurium, Clostridiumdifficile, Clostridium botulinum, Clostridium perfringens,Staphylococcus aureus, Escherichia coli (ETEC, EPEC, EHEC, EaggEC, UPECand EIEC), Campylobacter jejuni, Campylobacter coli, Campylobacter lari,Campylobacter fetus, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Listeria monocytogenes, Shigella, Streptococcus,Actinobacillus, Lactobacillus, Citrobacter, and Pseudomonas aeruginosa.

[0046] As used herein, “effective amount” refers to a dosage sufficientto have the desired effect.

[0047] Enterotoxigenic microorganisms cause a number of diseases anddisorders, including, for example, dysentery, gastroenteritis, typhoidfever, cholera, infectious hepatitis, poliomyelitis and diarrhea.Typically, normal motor propulsive activity, mucosal immunity and otherfactors limit the growth of organisms in the small intestine. However,when the stress of excess pathogens and toxins is too overwhelming forthe mucosal immune system, enterotoxigenic microorganisms colonize theGI tract, resulting in the diseases discussed above. It is of note that,in some cases, the enterotoxigenic microorganism is introduced into thehost GI tract by ingestion of contaminated food or water.

[0048] Described herein is a method of preparing a bioactive compounddelivery system. In some embodiments, the bioactive compound is arrangedto be delivered to the intestine, as discussed below. Also described arepharmaceutical compositions comprising at least one bioactive compoundwithin the delivery system. As discussed below, the delivery systemcomprises a matrix or lattice of at least one methacrylate polymer. Thebioactive compound may be any suitable compound. In a preferredembodiment, the bioactive compound is a compound which requires a nativesurface structure for proper interaction with its biological target.Examples of such compounds include antibodies, bacteriophage andreceptor ligands.

[0049] In one embodiment of the invention, there is provided apharmaceutical composition comprising a solution of methacrylic acidpolymers, a lyoprotectant and a bioactive compound. The pharmaceuticalcomposition may be lyophilized.

[0050] The lyoprotectant stabilizes the bioactive compound duringlyophylization. While not wishing to be bound or limited to a specifichypothesis, the inventors note that the lyoprotectant may act as anosmotic stabilizer. In view of this, compounds known in the art to actas osmotic stabilizer are suitable lyoprotectant. The lyoprotectant maybe for example but by no means limited to sucrose or glucose.

[0051] In some embodiments, the bioactive agent is a bacteriophagecapable of lysing at least one microorganism. In some embodiments, thebacteria is enterotoxigenic, although the pharmaceutical composition maybe used to treat any suitable bacterial infection. In these embodiments,the pharmaceutical composition contains an effective amount of thebacteriophage and is administered to an animal in need of suchtreatment. It is of note that bacteriophage capable of lysingenterotoxigenic bacteria are well known in the art.

[0052] In other embodiments, the bioactive agent is a pharmaceuticalagent, antibody, ligand or other such compound which requires a specificstructure for activity. As an illustrative example, the bioactivecompound may be a ligand for a specific cellular receptor and the ligandmay require a specific structure or conformation to interact with thereceptor. As will be appreciated by one of skill in the art, anybioactive compound known in the art for treating a specific disease ordisorder may be combined with the methacrylic polymer and lyoprotectantas discussed herein to conserve the structure or conformation of thebioactive molecule such that the bioactive molecule is more effective attreating the disease or disorder. As will be appreciated by one of skillin the art, exemplary diseases and disorders are not limited to the GItract but may be any disease or disorder wherein the therapeuticbioactive molecule or therapeutic bioactive agent requires maintenanceof a specific conformation or structure for activity.

[0053] Polymeric methacrylic acid copolmers (Eudragits) are typicallyapplied to pharmaceutical dosage forms at a pH below that where thepolymer enters solution, which is different for the various eudragitforms. The materials are therefore applied as a dispersion, rather thana solution. This has the benefit of creating an insoluble barrierresistant to acidic environments. It also permits the polymer to beapplied in a purely aqueous environment since the eudragits aretypically insoluble below a defined pH characteristic of the polymer.Polymethacrylic acid copolymers may also be applied in the protonatedaqueous insoluble form by dissolution in organic solvents, in which theyare soluble. This technique requires the use of organic solvents whichis generally undesirable.

[0054] The present invention provides methods and compositions forpreparing controlled release solid dosage forms containing viablebacteriophages. The preparations are prepared from polymethacrylic acidcopolymers using mild aqueous conditions. The present invention is notlimited to bacteriophages because it can be applied to preparingcontrolled release solid dosage forms of other complicated biologicmolecules and macromolecules. These include but are by no means limitedto, antibodies, receptor ligands, viruses, peptides, peptide fragments,enzymes, including digestive enzymes, DNA molecules, RNA molecules,growth factors, cytokines, bacteria and viruses, and the like.

[0055] Monolithic oral dosage formulations containing bacteriophages aremade by drying bacteriophages in an aqueous solution containing thereversibly pH-dependent soluble methacrylate polymer(s) (Eudragits;FDA-approved excipients) and a lyoprotectant, for example but by nomeans limited to sucrose and glucose. It is of note that while notwishing to be limited to a specific hypothesis, it is believed that thelyoprotectant acts as an osmotic stabilizer. Thus, other suitableosmotic stabilizing compounds known in the art may also be used aslyoprotectants. The formulations prepared in this manner using theingredients described contain highly viable bacteriophage that areprotected from the harsh environmental conditions in the stomach. Avariety of technologies, such as freeze drying and spray drying, may beused to prepare the dosage formulations. The formulations are suitablefor economical and practical treatment of a variety of bacterialinfections.

[0056] The ratio of polymer to lyoprotectant may vary from 200:1 to0.1:1.

[0057] Bacteriophages can be safely encapsulated in a matrix undergentle conditions in a manner that preserves their biological activityusing this method and compositions. Furthermore, the formulationsrelease the bacteriophages upon exposure to elevated pH conditionsencountered in the small intestine. As such, they represent a controlledrelease formulation.

[0058] These formulations have utility for the development ofbacteriophage therapy for other enteric and non-enteric bacterialinfections because they can be further modified using otherpharmaceutical production techniques to produce other usefulformulations. For example, the formulations may be further processed tomake other therapeutics by encapsulation in other polymeric substances,thereby producing formulations with different drug releasecharacteristics using means known in the art. For example, specificcarriers and carrier combinations known in the art may be selected basedon their properties and release characteristics in view of the intendeduse. Specifically, the carrier may be pH-sensitive, thermo-sensitive,thermo-gelling, arranged for sustained release or a quick burst. In someembodiments, carriers of different classes may be used in combinationfor multiple effects, for example, a quick burst followed by sustainedrelease.

[0059] In some embodiments, the above-described formulation may becombined with other compounds or compositions known in the art such thatthe is a pharmaceutical composition in the form of, for example, a pill,tablet, liquid, film or coating using means known in the art and asdiscussed below.

[0060] It is of note that the formulation discussed above may beprepared to be administered in a variety of ways, for example,topically, orally, intravenously, intramuscularly, subcutaneously,intraperitoneally, intranasally or by local or systemic intravascularinfusion using means known in the art and as discussed below.

[0061] All the formulations described above can be incorporated into avariety of edible materials, such as different foods and drinks, therebyincreasing their usefulness by providing vehicles of increasedpalatability that can be used to treat gastrointestinal tract infectionsin vertebrates and invertebrates. Furthermore, the formulations can beincorporated into wound dressings that can be used to treat bacterialinfections at poorly vascularized locations of the body.

[0062] It is of note that the compositions as described above may becombined with permeation enhancers known in the art for improvingdelivery. Examples of permeation enhancers include, but are by no meanslimited to those compounds described in U.S. Pat. Nos. 3,472,931;3,527,864; 3,896,238; 3,903,256; 3,952,099; 4,046,886; 4,130,643;4,130,667; 4,299,826; 4,335,115; 4,343,798; 4,379,454; 4,405,616;4,746,515; 4,788,062; 4,820,720; 4,863,738; 4,863,970; and 5,378,730;British Pat. No. 1,011,949; and Idson, “1975, J. Pharm. Sci. 64:901-924.

[0063] A pharmaceutical formulation that provides controlled release ofbacteriophage has not been previously reported. Benefits are that itprovides a formulation made using an aqueous system that does notrequire organic solvents. Furthermore it uses a versatile and economicalproduction method to produce a controlled release formulation containingstable and highly potent bacteriophage. Controlled release formulationsemploying methacrylate polymers used in the manner described here havenot been described elsewhere. The novel use is that the methacrylatesare used in aqueous solution, as opposed to the more common aqueousdispersion, and that enhanced-controlled release can be achieved duringa second step, namely acid treatment of dried monolithic matrix. Suchacid treatment may be achieved merely by exposure to acid in an acidicenvironment, such as the stomach. Specifically, the acid treatmentprotonates the carboxyl groups in the methacrylate polymer; these groupsare more protonated in dispersions, which are typically of a lower pHthan a solution of the same polymeric substance. The first step of theprocess, drying in the presence of methacrylate in solution in thepresence of lyoprotectant, provides a gentle condition under whichsensitive biological agents are dried (the bacteriophage are damaged informulation prepared with dispersions). As such, this process may alsobe used to make controlled release formulations of complex biologicalmolecules that are/may be used to treat non-infectious diseases.

[0064] The invention will now be described by way of examples; however,the examples are intended to be illustrative only.

EXAMPLE 1 Preparation of Bacteriophage rV5

[0065] Bacteriophage rV5 and wV8 were propagated in broth culture usingEscherichia coli strains sensitive to the bacteriophage according to themethods described in Sambrook and Russell (2001).

EXAMPLE 2 Preparation of Polymethyacrylate Copolymer Dispersions andSolutions

[0066] Eudragits are methacrylic acid copolymers used as excipients tocontrol the release of active ingredients from solid pharmaceuticaldosage formulations. These polymers have physiochemical properties, suchas dissolution within specified pH ranges, that permit protection andentrapment of active ingredients under certain harsh environmentalconditions, such as stomach. The polymers are typically used in twoways. To be exact, they are applied as thin layers from aqueousdispersions or organic solutions by spraying on cores containing drugs,or they are incorporated into monolithic forms as dry powders by directcompression techniques.

[0067] Substitution of the methacrylate polymer with varying numbers ofacidic or basic groups tailor release of the active ingredient indifferent regions of the gastrointestinal tract. For example, EudragitS100 is soluble only above approximately pH 7 and it facilitatescontrolled release in the colon. Eudragit L100 is soluble only above pH6 and it facilitates controlled release in the small intestine.Generally speaking, the protonated forms of Eudragit S100 and L100 areused in drug formulations because these forms are largely insolubleuntil the pH is elevated and the polymer is ionized by deprotonization.This necessitates using the polymers in one of two forms, which are asaqueous dispersions at a pH below the solution pH of the polymer, or asorganic solutions in which the polymer retains it protons. Dispersionmay be defined as a physiochemical system in which colloidal particlesare dispersed in a continuous phase of a different composition.Conversely, solution may be defined as a uniform mixture comprised of asolvent, usually a liquid, and a solute. Eudragit L100 and S100 arestructurally similar, but differ in the degree of substitution withacidic carboxyl groups on the polymer backbone. Dispersions andsolutions of Eudragits S100 and L100 may be prepared by varying the pHof the aqueous medium in which they are placed.

[0068] A dispersion of Eudragit L100 was made as follows:

[0069] Weigh out 110 g of Eudragit L100 solid (Rhom Pharma) and suspendthe particles in 730 ml of water.

[0070] Stir the mixture for 10 minutes at room temperature.

[0071] Add 10 N NaOH (Fisher Chemical Company) dropwise until a stablepH of approximately 5.7 is reached.

[0072] Store the material at room temperature.

[0073] The pH of the dispersion is below that required to dissolve theEudragit L100 polymer. The material has the appearance of a opaque whitelatex dispersion.

[0074] A solution of Eudragit S100 was made as follows:

[0075] Weigh out 110 g of Eudragit S100 solid (Rhom Pharma) and suspendthe particles in 730 ml of water.

[0076] Stir the mixture for 10 minutes at room temperature.

[0077] Add 10 N NaOH (Fisher Chemical Company) dropwise until a stablepH of approximately 7.1 is reached.

[0078] Store the material at room temperature.

[0079] The pH of the dispersion is above that required to dissolve theEudragit S100 polymer. The material has the appearance of a clear,colorless solution.

EXAMPLE 3 Stability of rV5 in Dried Formulations Containing a)Polymethacrylate Copolymer Eudragit L100, b) Sucrose, and c)Polymethacrylate Copolymer Eudragit L100 and Sucrose

[0080] Bacteriophage rV5 was added to different excipients mixturescomprised of 0-5% Eudragit L100 dispersion and 0-3% sucrose and theresulting material was dried by lyophilization after it was frozen at−70° C. The amount of active ingredient (ie. the number of viablebacteriophage rV5) in the excipients before freezing, after freezing,and after lyophilization was determined by titrating the samples on asuitable indicator strain using methods generally known to those skilledin the art. Before the dried formulations were titrated they weresolubilized with 50-100 mM carbonate/bicarbonate buffer forapproximately 10 minutes.

[0081] All the excipients maintained the viability of the bacteriophagebefore and after freezing, indicating that they did not directly damagethe bacteriophage in solution (Table 1). Zero, 2.5 and 5% sucrosemaintained the viability of the rV5 after lyophilization, provided noL100 was present. Eudragit L100 alone decreased the viability ofbacteriophage rV5 by 2-3 log₁₀ after lyophilization, indicating that italone damaged the bacteriophage greatly. Importantly, 5% sucrosetogether with 1.5 or 3% L100 maintained the viability of thebacteriophage. TABLE 1 Viability of rV5 in formulations containing a)polymethacrylate copolymer (Eudragit L100), b) sucrose, and c)polymethacrylate copolymer (Eudragit L100) and sucrose. BacteriophagerV5 (PFU/ml) Reconstituted Formulation Component Before after SucroseEudragit L100 Freezing After Freezing Lyophilization 0 0   2 × 10(8)   8× 10(8)   4 × 10(6) 0 0.375   4 × 10(7) 1.2 × 10(8)   6 × 10(3) 0 0.75  1 × 10(8)   2 × 10(6) 1.2 × 10(4) 0 1.5   4 × 10(7) 1.6 × 10(7) 1.4 ×10(4) 0 3   8 × 10(7)   2 × 10(7) 1.2 × 10(4) 2.5 0 2.6 × 10(7)   2 ×10(8)   1 × 10(7) 2.5 0.375   8 × 10(7) 1.2 × 10(9)   2 × 10(4) 2.5 0.75  2 × 10(7)   8 × 10(7)   6 × 10(4) 2.5 1.5   6 × 10(7)   8 × 10(7)   6× 10(4) 2.5 3 1.2 × 10(7)   4 × 10(7)   4 × 10(4) 5 0   4 × 10(7)   4 ×10(7) 1.2 × 10(6) 5 0.375   8 × 10(7) 1.2 × 10(8)   4 × 10(5) 5 0.75 1.2× 10(7) 1.2 × 10(8)   6 × 10(4) 5 1.5   6 × 10(7)   2 × 10(7)   6 ×10(5) 5 3   4 × 10(7)   4 × 10(7)   4 × 10(6)

[0082] These results were confirmed and extended in a relatedexperiment. In that experiment, 0, 0.125, 2.5 and 5% sucrose maintainedviability of the bacteriophage, provided the L100 was absent (Table 2).Three and 5% L100 decreased viability of the phage by 2 log₁₀. Adding2.5 or 5% sucrose to the 3 or 5% L100 maintained the viabilitycomparable to the sucrose only samples.

[0083] Whether this combination protected the bacteriophage from thedetrimental effects of acid was examined in part in Example 4. TABLE 2Viability of rV5 in formulations containing a) polymethacrylatecopolymer (Eudragit L100), b) sucrose, and c) polymethacrylate copolymer(Eudragit L100) and sucrose. Formulation Component (initial %, w/v)Bacteriophage rV5 (PFU/ml) in material Sucrose Eudragit L100reconstituted after lyophilization 0 0   8 × 10(6) 0 3   2 × 10(4) 0 5  6 × 10(4) 0.125 0   6 × 10(7) 0.125 3 1.2 × 10(5) 0.125 5 1.4 × 10(4)2.5 0   6 × 10(6) 2.5 3   2 × 10(6) 2.5 5 1.6 × 10(6) 5 0   4 × 10(7) 53   4 × 10(8) 5 5   2 × 10(7)

EXAMPLE 4 Stability of Bacteriophage rV5 in Dried FormulationsContaining Polymethacrylate Copolymer (Eudragit L100), Sucrose, andPolymethacrylate Copolymer (Eudragit L100) and Sucrose

[0084] Formulations containing bacteriophage rV5 were prepared asdescribed in Example 3 and these were left untreated or treated with 100mM HCl for 20 minutes. The resulting acid was neutralized withbicarbonate, which also dissolved the lyophilizate, and the number ofviable bacteriophage contained therein was determined as described inExample 3. As shown previously (Example 3), the formulations thatcontained sucrose alone and the formulations that contained sucrose andL100 maintained viability of the bacteriophage, and the formulationsthat contained L100 alone decreased the viability of the bacteriophage.Notably, the formulations that contained both L100 and sucrose togetherprotected the bacteriophage from acid treatment, whereas theformulations that contained L100 or sucrose alone did not. Theconculsion that can be drawn from this example is that polymethacrylicacid copolymer can protect the bacteriophage from harsh chemicalconditions, but only in the presence of another agent, for examplesucrose. TABLE 3 Protection of rV5 to acid treatment for 20 minutes byformulations containing a) polymethacrylate copolymer (Eudragit L100),b) sucrose, and c) polymethacrylate copolymer (Eudragit L100) andsucrose. Formulation Component Bacteriophage rV5 (PFU/ml) in (initial %,w/v) lyophilized material reconstituted Sucrose Eudragit L100 after 20min acid treatment 0 0 none detected 0 3 none detected 0 5 none detected0.125 0   1 × 10(3) 0.125 3   1 × 10(3) 0.125 5   3 × 10(4) 2.5 0 1.5 ×10(4) 2.5 3   3 × 10(4) 2.5 5   1 × 10(5) 5 0 2.5 × 10(4) 5 3   2 ×10(7) 5 5  25 × 10(6)

[0085] In a related experiment these results were confirmed andextended. In that experiment, formulations containing bacteriophage rV5and 0, 2.5, and 5% sucrose and 0, 3 and 5% L100 were treated overnightwith 100 mM HCl and then neutralized and solubilized with bicarbonatebuffer and titrated as described above to determine the number of viablebacteriophage remaining. Significantly, only the formulation containingsucrose and L100 afforded protection to the bacteriophage followingovernight exposure to acidic conditions (Table 4). TABLE 4 Protection ofrV5 to acid treatment overnight by formulations containing a)polymethacrylate copolymer (Eudragit L100), b) sucrose, and c)polymethacrylate copolymer (Eudragit L100) and sucrose. FormulationEudragit L100 (%, Sucrose Viability of Bacteriophage (PFU/ml) before (%,before Reconstituted without Reconstituted after drying) drying) acidtreatment overnight acid treatment 0 2.5 1.5 × 10(6) None 3 2.5   1 ×10(6) None 5 2.5 3.5 × 10(5) 5 × 10(5) 0 5   3 × 10(6) None 3 5   2 ×10(6) None 5 5 5.5 × 10(5) 5 × 10(4)

[0086] Whether this combination of materials entrapped thebacteriophage, or merely protected it in solution was examined inexperiments described in Example 5.

EXAMPLE 5 Retention of Bacteriophage in Acidic Environments byFormulations Containing Sucrose and S100

[0087] Dried formulations containing bacteriophage rV5 or wV8, 5%sucrose and 5-10% S100 were prepared according to Example 3 with S100solution prepared according to Example 2. These materials were treatedsequentially four times over a period of approximately 15 minutes with100 mM HCl. At the end of each treatment, the acid was removed,neutralized with bicarbonate buffer, then viable bacteriophage in thesamples were titrated according to Example 3. Furthermore, theformulation remaining at the end of the treatments was dissolved inbicarbonate buffer, and then the number of viable bacteriophage in itwas also determined by titration as described in Example 3. Nobacteriophages were liberated by the acid treatments and bacteriophageremained in the formulations after the acid treatments (Table 5, 6).TABLE 5 Controlled release of bacteriophage rV5 from formulation madeusing excipient containing 5% sucrose and 10% polymethacrylate S100.Bacteriophage content in sample Sample rV5 wV8 Original formulationbefore acid treatment   8 × 10(8) 1.5 × 10(7) Supernatant from acidtreatment 1 None none Supernatant from acid treatment 1 None noneSupernatant from acid treatment 1 None none Supernatant from acidtreatment 1 None none Remaining in formulation after acid 2.5 × 10(8)3.0 × 10(7) treatment PFU

[0088] TABLE 6 Controlled release of bacteriophage rV5 from formulationmade using excipient containing 5% sucrose and 5% polymethacrylate S100.Bacteriophage content in Sample Sample RV5 wV8 Original formulationbefore acid treatment 3.0 × 10(8) 1.5 × 10(8) Supernatant from acidtreatment 1 None none Supernatant from acid treatment 1 None noneSupernatant from acid treatment 1 None none Supernatant from acidtreatment 1 None none Remaining in formulation after acid 7.5 × 10(6)6.5 × 10(7) treatment PFU

[0089] A further experiment was conducted with formulations containingrV5 and wV8, S100 and sucrose as described above to further examine theability of the matrix to protect the bacteriophages. In that experiment,the formulations were exposed to acid for 20, 60, 120 minutes andovernight, and then the viable bacteriophage remaining was determined.Each matrix prevented inactivation of bacteriophages rV5 and wV8 duringthe overnight incubation with acid (Table 7). TABLE 7 Controlled releaseof bacteriophage rV5 and wV8 from formulations containingpolymethacrylate copolymer (Eudragit S100) and sucrose. Viability ofBacteriophage in Formulation of Different Compositions (PFU/ml) Time ofAcid 5% sucrose + 5% S100 5% sucrose + 10% S100 Treatment rV5 wV8 rV5wV8  0 minutes 3 × 10(8) 1.5 × 10(8)   8 × 10(8) 1.5 × 10(7)  20 minutes3 × 10(7) 5.5 × 10(7) 1.5 × 10(6) 2.5 × 10(7)  60 minutes 1 × 10(7)   1× 10(6)   1 × 10(7)   1 × 10(6) 120 minutes 5 × 10(7) 1.5 × 10(6) 7.5 ×10(7)   1 × 10(7) Overnight 7 × 10(6)   1 × 10(5)   1 × 10(7)   1 ×10(5)

[0090] Furthermore, an experiment was conducted to examine release ofbacteriophages rV5 and wV8 under mildly basic conditions. Driedformulations containing bacteriophage rV5 or wV8, 5% sucrose and 5-10%S100 were prepared according to Example 3 with S100 solution preparedaccording to Example 2. These materials were incubated with 10 mMphosphate buffered saline (pH 7.2) for 20, 60, 120 minutes andovernight, then centrifuged at 13,000×g, and then viable bacteriophagein the supernatant of the samples were titrated according to Example 3.Both rV5 and wV8 were rapidly released by the matrices in 20 minutes(Table 8). TABLE 8 Rapid release of rV5 and wV8 from formulationscontaining polymethacrylate copolymer (Eudragit S100) and sucrose.Release of Bacteriophage by Formulations of Different Compositions(PFU/ml) Time of PBS 5% sucrose + 5% S100 5% sucrose + 10% S100Treatment rV5 wV8 rV5 wV8  0 minutes 5 × 10(7) 1.5 × 10(8) 1.5 × 10(7)  1 × 10(7) (dissolved in bicarbonate buffer, pH 9.6)  20 minutes 1 ×10(8)   5 × 10(6)   5 × 10(7)   3 × 10(7)  60 minutes 2 × 10(8)   2 ×10(7) 2.5 × 10(8) 3.5 × 10(7) 120 minutes 5 × 10(7)   5 × 10(7) 1.5 ×10(7)   1 × 10(8) Overnight 5 × 10(7) 4.5 × 10(8)   1 × 10(8)   1 ×10(8)

[0091] These results show that formulations prepared in the manner ofExample 3 using solutions of polymethacylate copolymer trapmacromolecular biological material and protect it from acidicenvironments. Furthermore the formulations prepared in the manner ofExample 3 release the macromolecular material upon exposure toconditions that will result in dissolution of the entrapping polymer.

EXAMPLE 7 Spray Drying Bacteriophage in Polymethacrylate CopolymerSolution (Eudragit S100) Containing Sucrose

[0092] Liquid intermediaries containing bacteriophage rV5 were preparedcontaining 1.25, 2.5, and 5% sucrose and 2.5, 5, and 10% Eudragit S100,prepared as a solution according to Example 2 and then spray dried usingan inlet temperature 135 C and an outlet temperature 108 C. Theviability of bacteriophage in the resulting dried powder formulations,and the initial aqueous intermediary were determined as described inExample 3. Bacteriophage spray dried in all the excipients remainedviable (Table 8). TABLE 9 Viability of bacteriophage rV5 in formulationscontaining polymethacrylate copolymer (Eudragit S100) and sucroseprepared by spray drying. Bacteriophage Viability (PFU/ml) FormulationBefore Spray Drying After Spray Drying 2.5% S100, 1.25% 3.5 × 10(8) 4.1× 10(7) sucrose   5% S100, 2.5% sucrose 3.5 × 10(8) 1.5 × 10(7)  10%S100, 5% sucrose   5 × 10(8) 6.7 × 10(6)

[0093] While the preferred embodiments of the invention have beendescribed above, it will be recognized and understood that variousmodifications may be made therein, and the appended claims are intendedto cover all such modifications which may fall within the spirit andscope of the invention.

1. A pharmaceutical composition comprising: a methacrylic acid polymersolution or dispersion; a lyoprotectant; and a bioactive agent.
 2. Thepharmaceutical composition according to claim 1 wherein the compositionis lyophilized.
 3. The pharmaceutical composition according to claim 1wherein the lyoprotectant is glucose or sucrose.
 4. The pharmaceuticalcomposition according to claim 1 wherein the bioactive agent is abacteriophage.
 5. The pharmaceutical composition according to claim 1wherein the methacrylate polymer is Eudragit L100 or Eudragit S100.
 6. Amethod of preparing a pharmaceutical composition comprising: mixing amethacrylate polymer and water; adding a lyoprotectant and a bioactiveagent to the mixture; and drying the mixture.
 7. The method according toclaim 6 wherein the mixture is frozen prior to freeze-drying.
 8. Themethod according to claim 6 wherein the lyoprotectant is glucose orsucrose.
 9. The method according to claim 6 wherein the bioactive agentis a bacteriophage.
 10. The method according to claim 6 wherein themethacrylate polymer is Eudragit L100 or Eudragit S100.
 11. The methodaccording to claim 6 including adjusting the pH of the mixture.
 12. Amethod of treating a bacterial infection comprising: administering to ananimal in need of such treatment a therapeutic amount of a driedpharmaceutical composition comprising: a methacrylic acid polymer; alyoprotectant; and a bacteriophage.
 13. The method according to claim 12wherein the composition is lyophilized.
 14. The method according toclaim 12 wherein the composition is spray dried.
 15. The methodaccording to claim 12 wherein the lyoprotectant is glucose or sucrose.16. The method according to claim 12 wherein the bioactive agent is abacteriophage.
 17. The method according to claim 12 wherein themethacrylate polymer is Eudragit L100 or Eudragit S100.
 18. A method oftreating a bacterial infection comprising: administering to an animal inneed of such treatment a therapeutic amount of a dried pharmaceuticalcomposition comprising: a methacrylic acid polymer; a lyoprotectant; anda bioactive agent.
 19. The method according to claim 18 wherein thecomposition is lyophilized.
 20. The method according to claim 18 whereinthe composition is spray dried.
 21. The method according to claim 18wherein the lyoprotectant is glucose or sucrose.
 22. The methodaccording to claim 18 wherein the bioactive agent is a bacteriophage.23. The method according to claim 18 wherein the methacrylate polymer isEudragit L100 or Eudragit S100.
 24. A method of treating a diseasecondition comprising: administering to an animal in need of suchtreatment a therapeutic amount of a dried pharmaceutical compositioncomprising: a methacrylic acid polymer; a lyoprotectant; and a bioactiveagent.
 25. The method according to claim 24 wherein the composition islyophilized.
 26. The method according to claim 24 wherein thecomposition is spray dried.
 27. The method according to claim 24 whereinthe lyoprotectant is glucose or sucrose.
 28. The method according toclaim 24 wherein the bioactive agent is a bacteriophage.
 29. The methodaccording to claim 24 wherein the methacrylate polymer is Eudragit L100or Eudragit S100.